Expression of heterologous DNA sequences in a plant host is dependent upon the presence of an operably linked promoter that is functional within the plant host. Choice of the promoter sequence will determine when and where within the organism the heterologous DNA sequence is expressed. Where expression in specific tissues or organs is desired, tissue-preferred promoters may be used. Where gene expression in response to a stimulus is desired, inducible promoters are the regulatory element of choice. In contrast, where continuous expression is desired throughout the cells of a plant, constitutive promoters are utilized. Additional regulatory sequences upstream and/or downstream from the core promoter sequence may be included in the expression constructs of transformation vectors to bring about varying levels of expression of heterologous nucleotide sequences in a transgenic plant.
Frequently it is desirable to express a DNA sequence in particular tissues or organs of a plant. For example, increased resistance of a plant to infection by soil- and air-borne pathogens might be accomplished by genetic manipulation of the plant's genome to comprise a tissue-preferred promoter operably linked to a heterologous pathogen-resistance gene such that pathogen-resistance proteins are produced in the desired plant tissue.
Alternatively, it might be desirable to inhibit expression of a native DNA sequence within a plant's tissues to achieve a desired phenotype. In this case, such inhibition might be accomplished with transformation of the plant to comprise a tissue-preferred promoter operably linked to an antisense nucleotide sequence, such that expression of the antisense sequence produces an RNA transcript that interferes with translation of the mRNA of the native DNA sequence.
Thus far, the regulation of gene expression in plant roots has not been adequately studied despite the importance of the root to plant development. To some degree this is attributable to a lack of readily available, root-specific biochemical functions whose genes may be cloned, studied, and manipulated. Genetically altering plants through the use of genetic engineering techniques and thus producing a plant with useful traits requires the availability of a variety of promoters. An accumulation of promoters would enable the investigator to design recombinant DNA molecules that are capable of being expressed at desired levels and cellular locales. Therefore, a collection of tissue-preferred promoters would allow for a new trait to be expressed in the desired tissue. Several genes have been described by Takahashi et al. (1991) Plant J. 1:327–332; Takahashi et al. (1990) Proc. Natl. Acad. Sci. USA 87:8013–8016; Hertig et al. (1991) Plant Mol Biol. 16:171–174; Xu et al. (1995) Plant Mol Biol. 27:237–248; Capone et al. (1994) Plant Mol Biol. 25:681–691; Masuda et al. (1999) Plant Cell Physiol. 40(11):1177–81; Luschnig et al. (1998) Genes Dev. 12(14):2175–87; Goddemeier et al. (1998) Plant Mol Biol. 36(5):799–802; and Yamamoto et al. (1991) Plant Cell. 3(4):371–82 to express preferentially in plant root tissues.
Metallothioneins (MT's) are proteins or polypeptides that bind and sequester ionic forms of certain metals in plant and animal tissues. Examples of such metals include copper, zinc, cadmium, mercury, gold, silver, cobalt, nickel and bismuth. The specific metals sequestered by MT's vary for the structurally distinct proteins/polypeptides occurring in different organisms. Plants appear to contain a diversity of metal-binding MT's with the potential to perform distinct roles in the metabolism of different metal ions. In plants, MT's are suggested to have roles in metal accumulation, metal intoxication, and embryogenesis (Thomas et al. (2003) Biotechnol. Prog. 19:273–280; Dong and Dunstan (1996) Planta 199:459–466; Kawashima et al. (1992) Eur. J. Biochem. 209:971–976).
Typically, MT's and MT-like proteins are Cys-rich proteins that are characterized by the presence of Cys-Xaa-Cys motifs suggesting the capability of binding metal ions. Further categories of MT-like proteins have been proposed on the basis of the predicted locations of Cys residues and have been designated types 1 and 2. In type 1 there are exclusively Cys-Xaa-Cys motifs, whereas in type 2 there is a Cys-Cys and a Cys-Xaa-Xaa-Cys pair within the N-terminal domain. The type 1 motif has been implicated in the binding and sequestration of copper (Murphy et al. (1997) Plant Physiol. 113:1293–1301 and Carr et al. (2002) J. Biol. Chem. 277:31237–31242).
Several metallothionein-like plant genes have been isolated, including those from pea, maize, barley, Mimulus (monkeyflower), soybean, castor bean and Arabidopsis. See Robinson et al. (1993) Biochem J. 295: 1–10. Sequences expressed in roots have been reported to be isolated from pea, as described in Evans et al. (1990) FEBS Lett 262:29–32. A maize root MT gene has been described in U.S. Pat. No. 5,466,785; though this sequence is also expressed in leaves, pith and seed, as described in de Framond (1991) FEBS Lett 290:103–106.
Thus, isolation and characterization of tissue-preferred, particularly root-preferred, promoters that can serve as regulatory regions for expression of heterologous nucleotide sequences of interest in a tissue-preferred manner are needed for genetic manipulation of plants.